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Endocytosis of Tight Junction Proteins Following RSV Infection Leads to Airway Epithelial Barrier Dysfunction
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A2859 - Endocytosis of Tight Junction Proteins Following RSV Infection Leads to Airway Epithelial Barrier Dysfunction
Author Block: D. Linfield1, A. I. Ivanov2, G. Piedimonte2, F. Rezaee3; 1Cleveland Clinic Lerner College of Medicine, Cleveland, OH, United States, 2Pediatrics, Cleveland Clinic, Cleveland, OH, United States, 3Center for Pediatric Research, Cleveland Clinic, Cleveland, OH, United States.
RATIONALE:
Respiratory syncytial virus (RSV) is a major cause of hospitalization for infants and young children worldwide. To date, RSV treatment largely remains supportive care, as no vaccine is currently available. Epithelial apical junctional complex (AJC) of airway epithelial cells creates a barrier against airborne threats. Recent studies suggest that RSV leads to AJC dysfunction, without changes in AJC protein levels, suggesting that the proteins travel elsewhere in the cell. Endocytosis plays a fundamental role in the regulation of barrier structure and function. In this study, we aimed to elucidate if RSV infection disrupts airway epithelial barrier by triggering endocytosis of AJC proteins.
METHODS:
Confluent monolayers of human bronchial epithelial cells (16HBE14o) were infected with RSV strain A2 at a multiplicity of infection (MOI) of 1 for 48 hours. We focused on the three main mechanisms of endocytosis, which are: 1. clathrin-mediated (CDE), 2. caveolae or lipid raft-mediated (CavME), and 3. macropinocytosis. Cells were treated with selective pharmacological inhibitors for each endocytic pathway. AJC function was evaluated using transepithelial electrical resistance (TEER). Immunofluorescence staining and confocal microscopy following an internalization assay were used to study selective markers of the endocytosis pathways: FITC-labeled transferrin for CDE, FITC-labeled cholera toxin for lipid raft-mediated endocytosis, and FITC-labeled dextran for macropinocytosis.
RESULTS:
TEER readings decreased for all RSV-infected cells, in comparison to non-infected cells. In comparison to non-infected cells, there appeared to be increases in amounts of both dextran and transferrin within the cytoplasm of the cells following RSV infection, while cholera toxin levels remained constant. Furthermore, RSV-induced AJC disassembly was attenuated by selective pharmacological inhibition of macropinocytosis and CDE.
CONCLUSIONS:
These findings suggest that RSV infection induces AJC disassembly by increasing clathrin-mediated endocytosis and macropinocytosis of junctional proteins. This will result in decreased amount of AJC proteins at the intracellular junctions, leading to disruption of the airway epithelial barrier.
Author Block: D. Linfield1, A. I. Ivanov2, G. Piedimonte2, F. Rezaee3; 1Cleveland Clinic Lerner College of Medicine, Cleveland, OH, United States, 2Pediatrics, Cleveland Clinic, Cleveland, OH, United States, 3Center for Pediatric Research, Cleveland Clinic, Cleveland, OH, United States.
RATIONALE:
Respiratory syncytial virus (RSV) is a major cause of hospitalization for infants and young children worldwide. To date, RSV treatment largely remains supportive care, as no vaccine is currently available. Epithelial apical junctional complex (AJC) of airway epithelial cells creates a barrier against airborne threats. Recent studies suggest that RSV leads to AJC dysfunction, without changes in AJC protein levels, suggesting that the proteins travel elsewhere in the cell. Endocytosis plays a fundamental role in the regulation of barrier structure and function. In this study, we aimed to elucidate if RSV infection disrupts airway epithelial barrier by triggering endocytosis of AJC proteins.
METHODS:
Confluent monolayers of human bronchial epithelial cells (16HBE14o) were infected with RSV strain A2 at a multiplicity of infection (MOI) of 1 for 48 hours. We focused on the three main mechanisms of endocytosis, which are: 1. clathrin-mediated (CDE), 2. caveolae or lipid raft-mediated (CavME), and 3. macropinocytosis. Cells were treated with selective pharmacological inhibitors for each endocytic pathway. AJC function was evaluated using transepithelial electrical resistance (TEER). Immunofluorescence staining and confocal microscopy following an internalization assay were used to study selective markers of the endocytosis pathways: FITC-labeled transferrin for CDE, FITC-labeled cholera toxin for lipid raft-mediated endocytosis, and FITC-labeled dextran for macropinocytosis.
RESULTS:
TEER readings decreased for all RSV-infected cells, in comparison to non-infected cells. In comparison to non-infected cells, there appeared to be increases in amounts of both dextran and transferrin within the cytoplasm of the cells following RSV infection, while cholera toxin levels remained constant. Furthermore, RSV-induced AJC disassembly was attenuated by selective pharmacological inhibition of macropinocytosis and CDE.
CONCLUSIONS:
These findings suggest that RSV infection induces AJC disassembly by increasing clathrin-mediated endocytosis and macropinocytosis of junctional proteins. This will result in decreased amount of AJC proteins at the intracellular junctions, leading to disruption of the airway epithelial barrier.
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